As used herein, the term "ligand-receptor" assay refers to an assay for an analyte which may be detected by the formation of a complex between a ligand and another substance capable of specific interaction with that ligand, i.e., ligand receptor. The ligand may be the analyte itself or a substance which, if detected, can be used to infer the presence of the analyte in a sample. In the context of the present invention, the term "ligand", includes haptens, hormones, antigens, antibodies, deoxyribonucleic acid (DNA), ribonucleic acids (RNA), metabolites of the aforementioned materials and other substances of either natural or synthetic origin which may be of diagnostic interest and have a specific binding partner therefor, i.e., the ligand receptor in the ligand-receptor assay. The term "ligand receptor" includes materials for which there is a specific binding partner, i.e., the ligand of the ligand-receptor assay. Those skilled in the art will appreciate that the analyte of interest, a member of a specific binding pair, may be either ligand or ligand receptor depending upon assay design.
Ligand-receptor assays are generally useful for the in-vitro determination of the presence and/or concentration of ligands in body fluids, food products, and environmental samples. For example, the determination of specific hormones, proteins, therapeutic drugs, and toxins in human body fluids has significantly improved the ability of medical practice to diagnose and minister to the human condition. There is a continuing need for simple, rapid, non-instrumental assays for the qualitative and semi-quantitative determination of such ligands in a sample. This need for simple, rapid methods entails a concomitant requirement for assay devices to complement such assay methods. Furthermore, in many situations, such assays methods need to be simple enough to be performed and interpreted by non-technical users without the requirement of costly and complex apparatus suitable for use only in a laboratory setting by highly skilled personnel.
Ligand-receptor assays rely on the binding of ligands by receptors to determine the concentration of ligands in a sample. Ligand-receptor assays can be characterized as either competitive or non-competitive. Non-competitive assays generally utilize receptors in substantial excess over the amount of ligand to be determined. Sandwich assays, in which the ligand is detected by binding to two receptors, one receptor labeled to permit detection and a second receptor typically bound to a solid phase to facilitate separation of bound from unbound reagents, such as unbound labeled first receptor, are examples of noncompetitive ligand-receptor assays. Proteins, hormones and deoxyribonucleic acid (DNA) are examples of ligands commonly detected using non-competitive assays. Competitive assays generally involve ligand from the sample, a ligand analogue labeled to permit detection, and the competition of these species for a limited number of ligand receptor binding sites. Examples of ligands which are commonly measured by competitive ligand-receptor assays include haptens, hormones and proteins. Antibodies that can bind these classes of ligands are frequently used in both non-competitive and competitive assays as the ligand receptors.
Ligand-receptor assays can be further described as being either homogeneous or heterogeneous. In homogeneous assays, all of the reactants participating in the reaction are admixed and the quantity of ligand is determined by its effect on the binding events involving the labeled species. The signal observed is modulated by the extent of this binding and can be related to the amount of ligand in the sample. U.S. Pat. No. 3,817,837 describes such a homogeneous, competitive immunoassay in which the labeled ligand analogue is a ligand-enzyme conjugate and the ligand receptor is an antibody capable of binding to either the ligand or the ligand analogue. The binding of the antibody to the ligand-enzyme conjugate decreases the activity of the enzyme relative to the activity observed when the ligand-enzyme conjugate is in the unbound state. Due to competition between unbound ligand and ligand-enzyme conjugate for antibody binding sites, as the ligand concentration increases the amount of free ligand-enzyme conjugate increases and thereby increases the observed signal. The product of the enzyme reaction may then be measured kinetically using a spectrophotometer.
Heterogeneous ligand-receptor assays require a separation of bound labeled ligand receptor or labeled ligand analogue from the free labeled ligand receptor or labeled ligand analogue and a subsequent measurement of either the bound or the free fraction. Methods for performing such heterogeneous, competitive assays are described in U.S. Pat. Nos. 3,654,090, 4,298,685, and 4,506,009; such a non-competitive assay is described in U.S. Pat. No. 4,376,110.
The need for ligand-receptor assays that can be performed without the use of instrumentation has led to the development of assay devices that can be visually interpreted. U.S. Pat. Nos. 4,125,372, 4,200,690, 4,246,339, 4,366,241, 4,446,232, 4,477,576, 4,496,654, 4,632,901, 4,727,019, and 4,740,468 describe devices and methods for heterogeneous, ligand-receptor assays that can develop colored responses to permit visual interpretation of the results.
Among the first devices developed for ligand-receptor assays were simple dipstick type devices designed for contacting a porous material such as a membrane with both the sample and labeled reagents via immersion allowing appropriate reagent incubations to occur and then separating the free from the bound label using a wash step. Such devices are described in U.S. Pat. Nos. 3,715,192, 4,200,690, and 4,168,146 and EPO Appl. Nos. 0 032 286 and 0 063 810. A common distinguishing feature of devices constructed in a dipstick format is the absence of a fluid receiving zone within the device for containing the sample, liquid reagents and wash solutions after the performance of the sample and reagent incubations and the separation of free from bound label. The lack of such a fluid receiving zone precludes characterization of such a dipstick device as self-contained, given that some external fluid receptor must be provided to capture used sample, unbound labeled reagents and spent wash fluid.
A class of devices which constitute an improvement over the simple dipstick construct is the immunochromatographic test strip device. This class of device generally exhibits improved sensitivity in ligand detection relative to that of simple dipstick devices by virtue of the ligand concentrating effect achieved by the flow of sample containing the ligand past an immobilized ligand receptor zone. Such devices also provide a limited fluid receiving zone for fluids used in the performance of the assay. A fluid receiving zone is created by increasing the length of the porous member to provide a suitable amount of total void volume capacity. Such devices are described in U.S. Pat. Nos. 4,094,647, 4,235,601, 4,361,537, 4,366,241, 4,435,504, 4,624,929, 4,740,468, 4,756,828, and 4,757,004; EPO. Appl. Nos. 0 267 006, 0 271 204, and 0 299 428; and PCT Appl. No. US86/0668. Even though such immunochromatographic devices do include a limited fluid receiving zone, they do not enable an efficient free/bound label separation, since the rate of separation is slow and limited by the rate at which fluid travels along the length of the porous member. Some immunochromatographic devices are so limited by the capacity of their fluid zone that no free/bound label separation can be performed; such devices rely upon the increase in concentration of label at the immobilized ligand receptor zone to distinguish bound from free label. A need exists for a device that is both efficient and rapid in performing separation of the free from the bound label in an assay.
A specialized form of an immunochromatographic device is employed in the method of radial partition immunoassay. In this assay method, the sample and labeled reagents are carefully applied to the immobilized receptor zone in the center of the porous material. The wash fluid is then also carefully applied to the immobilized receptor zone and the unbound label flows radially away from the central immobilized receptor zone. Radial partition immunoassay devices like the aforementioned immunochromatographic devices require that the volume of wash fluid be less than the total void volume of the porous member containing the ligand receptor since it is the void volume of the porous member in excess of the volume of the sample which provides the necessary additional fluid capacity. Such radial partition immunoassay devices are described in U.S. Pat. Nos. 4,517,288, 4,670,381, 4,752,562, 4,774,174, and 4,786,606. Devices used for radial partition immunoassay are not generally suitable for the detection of a multiplicity of ligands. The usable ligand detection zone necessarily must be relatively small and constrained since the physical separation of free and bound labeled species is strictly limited by the dimensions of the device and the total fluid capacity of the porous member.
Immunochromatographic and radial partition immunoassay devices depend primarily on horizontal separation, i.e., along or within the plane of the porous member, of the free and bound labeled species in order to achieve acceptable physical separation of the free from the bound labeled reagents. A separate class of devices utilizes flow of fluid in a direction which is primarily transverse to the plane of the porous member. Devices which operate in this manner may be generally referred to as "flow- through" devices. The absorbent material which constitutes the fluid receiving zone in these devices can either be in non-continuous contact with the porous member containing immobilized receptor as described in U.S. Pat. Nos. 3,888,629 and 4,246,339 or in continuous contact with the porous member as described in U.S. Pat. Nos. 4,366,241, 4,446,232, 4,632,901 and 4,727,019, and in EPO. Appl. No. 0 28.1 201. Devices in which the absorber is not in continuous contact with the porous member such as described in U.S. Pat. Nos. 3,888,629 and 4,246,339 allow the contact of the solutions containing sample and/or labeled reagents with the porous member to occur prior to permitting flow of the labeled reagent solution into the fluid absorbent. Since the contact is not continuous between the absorber and the porous member, the volume of fluid needed to ensure that the porous member is completely saturated is only the void volume of the porous member. Such non-continuous contact devices are inherently more efficient at utilization of sample and labeled reagents and thus-by this measure are more cost-effective than are continuous contact flow-through devices such as those described in U.S. Pat. Nos. 4,446,232, 4,632,901 and 4,727,019. The non-continuous contact flow-through devices however, have the disadvantage that a physical motion is required by the assayist to bring the separated absorber into contact with the porous member and to thereby enable the flow of fluid needed for separation of free label from bound label. The requirement of direct mechanical intervention is not desirable from the perspective of ease of use by non-trained users, as it introduces a step which may be subject to error. The continuous contact flow-through devices eliminate the need for active intercession by the user to complete the fluid contact between the absorber and the porous member, but are less efficient in the utilization of costly labeled reagents. The flow characteristics of such devices are optimized such that fluid flow in the direction transverse to the plane of the porous member is preferred. Thus, a reagent volume substantially greater than the void volume of the porous member is required to ensure that the entire porous member has been contacted with the solution containing reagents. Since neither the non-continuous nor the continuous contact flow-through devices described in the prior art are capable of providing a device which exhibits the characteristics both of efficient use of labeled reagents and of avoiding the need for an additional mechanical intercession step, there remains an unmet need for a device with such attributes.
The inventive devices herein described are not limited to either a flow through or an immunochromatographic method but rather may be modified to achieve the benefits of both by, for example, controlling the placement of the sample or the design and placement of the porous and non-absorbent members. In preferred embodiments reagent flow is primarily tangential to the porous membrane while washing reagent flow is primarily transverse to the membrane and then into the network of capillary channels. These features distinguish this invention over the flow through and immunochromatographic devices of the prior art.
Control of the rate and path of fluid flow in an assay device can be of paramount importance. To achieve this end, a number of devices have been described in the prior art which use surfaces with specifically arranged geometric elements to control the path and the rate of fluid flow. Devices such as are described in U.S. Pat. Nos. 3,690,836 and 4,426,451 and EPO. Appl. No. 0 034 049 utilize an arrangement in which a porous member is placed between smooth surfaced planar sheets of a non-absorbent material in order to contain a fluid within the porous material. Devices such as are described in U.S. Pat. Nos. 4,233,029 and 4,310,399 use geometric arrangements of capillary channels to modulate the flow of fluid, such that fluid is directed to flow in regular geometric patterns and at controlled rates. A device for controlling the delivery of fluid a porous member using a textured surface possessing a surface capillary network is described in EPO. Appl. No. 0 239 174. while the devices described are suitable for control of fluid flow, they fail to control fluid flow through a porous member such that assay devices can be constructed to make efficient use of sample and labeled reagents and to contain a suitable fluid receiving zone for use in achieving a rapid and efficient separation of free from bound labeled species in an assay. Thus remains a need which has been unmet by any of the aforementioned architecture-controlled flow devices.
A preferred device for performing ligand-receptor assays should not impose the need for mechanical intercession on the assay procedure because this may introduce operator error. The inventive devices herein described and claimed are efficient in their use of sample and costly reagents and provide an adequate fluid receiving zone for all liquid reagents, particularly those of the free/bound separation step in an assay. The devices are capable of supporting ligand-receptor assays directed to simultaneous detection of a multiplicity of target ligands and they may be used in ligand-receptor assay formats which are analogous both to those of flow-through assays and to those of immunochromatographic assays.
One advantage of the devices herein described is the efficient use of reagents while incurring a minimum number of steps in the assay protocol. The device allows one to use a large porous membrane and to cover it with multiple ligand receptor zones, because it ensures that the sample will flow over and cover the entire membrane. This is accomplished without the need for either large sample volumes or mechanical action. Another advantage of this invention is the non-absorbent member. When an excess volume of fluid is added, the network of capillary channels formed by the contact of the porous member and the nonabsorbent member ensures washing efficiency by directing flow away from the porous member, thereby assuring good separation of free from bound labeled conjugate. In one embodiment the inventive device can be employed in assays using flow-through methods. In another embodiment the described device can perform assays using immunochromatographic methods. Further, the device of the present invention efficiently performs the task of separating free labeled species from bound labeled species, a pivotal requirement for heterogeneous ligand-receptor assay methods.